Ethylenediamine platinum(II) and 1,2-diamino-cyclohexane platinum(II) pyrophosphate complexes

ABSTRACT

Ethylenediamine platinum(II) and 1,2-diaminocyclohexane platinum(II) pyrophosphate complexes are prepared by reaction of the appropriate diaquo platinum(II) salt with a pyrophosphate salt. These complexes possess pronounced anti-tumor activity and low toxicity and thus have high therapeutic indices.

This invention is concerned with certain complexes of platinum(II)containing a pyrophosphate moiety and a bidentate amine moiety. Moreparticularly, this invention is concerned with amine platinum(II)pyrophosphate complexes where the amine moiety is an ethylenediamine ora 1,2-diaminocyclohexane moiety. These compounds have the generalformula [{Pt(A)}₂ P₂ O₇ ], where A is either an ethylenediamine or a1,2-diaminocyclohexane moiety. These complexes are characterized bypronounced activity against tumors combined with low animal toxicity.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the infrared spectrum of the ethylenediamine platinum(II)pyrophosphate complex of Example 1.

FIG. 2 shows the infrared spectrum of the 1,2-diaminocyclohexaneplatinum(II) pyrophosphate complex of Example 2.

BACKGROUND

Rosenberg et al reported the discovery that certain platinumcoordination compounds are of interest as potential antitumor agents.(Rosenberg et al, "Platinum Compounds: A new Class of Potent Anti-TumorAgents," Nature, Vol. 222 (Apr. 26, 1969), pp. 385-86). Since then,considerable effort has been expended to evaluate various classes ofcoordination complexes for similar activity. See, e.g., M. J. Cleare,"Transition Metal Complexes in Cancer Chemotherapy," CoordinationChemistry Reviews, 12 (1974), pp. 349-405. Cis-diammineplatinumpyrophosphate complexes of the empirical formula [{Pt(NH₃)₂ }₂ P₂ O_(7])have been reported; however, they have only marginal activity. (Cleareet al, "Studies of the Antitumor Activity of Group VIII Transition MetalComplexes. Part I, Platinum(II) Complexes," Bioinorganic Chemistry, 2,187-210 (1973) at p. 199.)

A blue, water soluble, orthophosphate complex of1,2-diaminocyclohexaneplatinum(II) containing 45.85% platinum (Pt) hasbeen reported to show anti-tumor activity (G. R. Gale et al,"Preparation and Anti-tumor Evaluation of Water-Soluble Derivatives ofDichloro-1,2-diaminocyclohexane platinum", Cancer Treatment Reports 61pp. 1519-1525 (1977).

SUMMARY OF THE INVENTION

In accordance with the present invention, there are provided novelethylenediamine platinum(II) and 1,2-diaminocyclohexane platinum(II)pyrophosphate complexes which, unlike the known pyrophosphate complex,have exhibited pronounced anti-tumor activity. In addition, they havelow mammalian toxicity. As a consequence, in addition to their markedactivity, the complexes of this invention have a favorable therapeuticindex.

The complexes of this invention may be represented by the generalformula [{Pt(A)}₂ P₂ O₇ ]. Thus, pyrophosphate comprises a tetravalentmoiety which is represented by the formula P₂ O₇ ⁻⁴.

The remainder of the platinum(II) complexes of this invention comprisesa Pt(II)A moiety, wherein A is a bidentate diamine moiety. Thus, A maybe represented by the general formula: ##STR1## wherein each of R¹ -R⁶,when taken separately, is hydrogen or lower alkyl; and R³ and R⁴, whentaken together, form the divalent radical: ##STR2## wherein each of R⁷-R¹⁰, when taken separately, is hydrogen or lower alkyl, giving a1,2-diaminocyclohexane with the structure: By the term "lower alkyl", asemployed herein, is meant a linear or branched chain alkyl group of from1 to about 6 carbons, and preferably from 1 to about 3 carbons, such asmethyl, ethyl and propyl. Preferred are diamines where at least five ofsaid R¹ -R⁶ are hydrogen, such as ethylenediamine andpropylene-1,2-diamine. Also preferred are the diamines where at leastthree of R¹, R², R⁵ and R⁶ are hydrogen, and R³ and R⁴, when takentogether, are replaced by the ##STR3## radical wherein, each of R⁷ -R¹⁰is hydrogen, such as 1,2-diaminocyclohexane.

The complexes of this invention are prepared by reacting adiaquodiamineplatinum(II) salt with a pyrophosphate salt in an aqueousmedium. The diaquodiamineplatinum(II) salt may be represented by theformula:

    [Pt(A)(H.sub.2 O).sub.2 ].sup.+2 (X.sup.-(2-n)).sub.1+n

wherein X is an inorganic anion and n is 0 or 1. Suitable anions arethose which are stable in acid media and which do not affect pH; theyinclude sulfate, nitrate and perchlorate, although nitrate is preferred.Anions having a greater complexing ability than water or pyrophosphate,such as chloride, iodide and bromide are not suitable.

The diaquo salt is formed by the stoichiometric reaction of theappropriate dichlorodiamineplatinum(II) analogue with a silver salt,preferably silver nitrate, in an aqueous medium at room temperature. Thediaquo salt is unstable in solution, but may be converted to stablesolid [Pt(A)(OH)]₂ (X)₂ by reaction with one gram mole of base per gramatom of platinum. This dimeric complex may be reconverted to monomerwith acid or used directly in the preparation of phosphate compounds.

The pyrophosphate salts which are employed are water soluble salts,preferably alkali metal pyrophosphate salts such as tetrasodiumpyrophosphate.

The pyrophosphate complexes of this invention are prepared by thereaction of equimolar amounts of a diaquoplatinum(II) salt with thegeneral formula [Pt(A)(H₂ O)₂ ]X₂ and a pyrophosphate salt, such astetrasodium pyrophosphate in aqueous media. The reaction proceedsaccording to the following equation:

    [Pt(A)(H.sub.2 O).sub.2 ]X.sub.2 +P.sub.2 O.sub.7.sup.-4 →[{Pt(A)}.sub.2 P.sub.2 O.sub.7 ]

Upon the first mixing of the reactants, a white precipitate forms,which, when A is an ethylenediamine moiety, may be converted to thedesired yellow end product by one of the following methods: (1)recrystallization of the white solid from boiling water; or (2) brieflyboiling the initial reaction mixture with the yellow product separatingupon cooling. Addition of nitric acid (less than one gram mole nitricacid per gram atom platinum) to the mixture while it is boiling willincrease the yield of the yellow product. When A is a1,2-diaminocyclohexane moiety, the yellow end product is obtained bydrying the while solid in a vacuum desiccator at room temperature for 24hours.

The complexes of this invention are especially useful in tumorchemotherapy, having been found active against sarcoma 180 ascites,lymphoid leukemia L1210, lymphocitic leukemia P388 and B16 melanoma inmice. The complex is administered intraperitoneally as a slurry with asuspending agent such as Klucel (hydroxypropyl cellulose). The slurrymay contain other components, such as physiologically acceptable salts,other medicaments, etc. The dosage level required for anti-tumoractivity is not narrowly critical, and indeed it is a feature of thecomplexes of this invention that, because of their relatively lowtoxicity, they may be administered over a wide dosage range. In mice,dosages from about 5 to about 30 mg/kg of body weight have been foundeffective.

The following examples are illustrative. In the examples, the symbol"en" designates the ethylenediamine moiety and the symbol "DAC"designates the 1,2-diaminocyclohexane moiety.

EXAMPLE 1 Synthesis of Ethylenediamine Platinum(II) PyrophosphateComplex

A 13.4 gram portion of solid Na₄ P₂ O₇.10H₂ O was added to 120 ml of a0.25 M solution of [Pt(en)(H₂ O)₂ ](NO₃)₂ so that the pyrophosphate toplatinum ratio was about 1. A milky precipitate was produced whichdissolved upon heating the mixture to boiling. The addition of 3 ml of 9M nitric acid immediately precipitated a yellow crystalline solid.Cooling the mixture to about 5° C. and storing it at that temperaturefor about 6 or 7 hours produced more of the yellow solid. This yellowproduct was filtered, washed with water and ethanol, and air dried,yielding 4.59 grams (or 44.7% based on Pt analysis) of the aboveidentified complex. The complex appeared to be completely air stable.

Elemental analysis of the complex gave the following results:

    ______________________________________                                        Analysis [{Pt(C.sub.2 H.sub.8 N.sub.2)}.sub.2 P.sub.2 O.sub.7 ]               % Pt         % C      % H      % N    % P                                     ______________________________________                                        Calculated:                                                                           57.02    7.02     2.36   8.19   9.05                                  Found:  57.21    7.11     2.45   7.96   8.95                                  ______________________________________                                    

The infrared spectrum for this complex is attached as FIG. 1. The bandsat 1200, 1150, 1050, 1010 and 960 cm⁻¹ are assigned to the coordinatedpyrophosphate moiety. The band at 1575 cm⁻¹ is assigned toδ_(NH).sbsb.2. The band at 860 cm⁻¹ is assigned to ρ_(NH).sbsb.2. Theband at 560 cm⁻¹ is assigned to νPt-N. The bands at 450 cm⁻¹ and 500cm⁻¹ are assigned to νPt-O.

EXAMPLE 2 Synthesis of 1,2-Diaminocyclohexane Platinum(II) PyrophosphateComplex

A solution made of 5.09 grams Na₄ P₂ O₇.10H₂ O dissolved in 40 ml waterwas slowly added to 200 ml of a 0.057 M solution of [Pt(DAC)(H₂ O)₂](NO₃)₂. A white precipitate began to form almost immediately uponaddition of the pyrophosphate solution. The reaction mixture was stirredovernight, then filtered. The resulting solid filtrate was washed threetimes with cold water to yield the above-identified complex as anoff-white solid that had a very slight green tinge when wet. The complexwas air and vacuum-dried, yielding 0.302 grams of yellow complex whichwas similar in appearance to the complex of Example 1. The yellowcomplex was not air stable and was reconverted to the white materialupon standing in air for 24 hours.

Elemental analysis of the complex gave the following results:

    ______________________________________                                        Analysis: [{Pt(C.sub.6 H.sub.14 N.sub.2)}.sub.2 P.sub.2 O.sub.7 ]             % Pt         % C      % H      % N    % P                                     ______________________________________                                        Calculated:                                                                           49.23    18.19    3.56   7.07   7.82                                  Found:  52.34    17.93    3.58   6.83   8.10                                  ______________________________________                                    

The infrared spectrum is attached as FIG. 2. The band assignments appearto be the same as those for Example 1.

EXAMPLE 3 Evaluation of Anti-Tumor Activity in the Mouse S180a System

The ethylenediamine platinum(II) pyrophosphate complex of Example 1 andthe 1,2-diaminocyclohexane platinum(II) pyrophosphate complex of Example2 were tested for anti-tumor activity against S180 ascites in femaleSwiss white mice by the following procedure:

CFW mice, averaging 20 grams, were immediately inspected weighed, andthen placed in newly prepared cages (6 mice/cage or 1 set). On day 0 themice were inoculated with 0.2 ml of a freshly prepared saline suspension(0.15 M NaCl) containing 1×10⁷ tumor cells/ml or a total of 2×10⁶ cells.This inoculum was freshly prepared using "transfer" mice which had beeninjected with tumor cells the previous week, and was the end-product ofa series of steps which involved (1) the removal of the cells from theperitoneal cavity of the sacrificed transfer mouse, (2) alternatecentrifugation-washing (2-3 times with cold saline) to remove occasionalblood and other undesirable components, and (3) dilution (1:3) of thepacked cell volume with saline (the final centriguation being carriedout at 1000 rpm for two minutes). A cell count was made (in duplicate)on a 100-fold dilution of this 1:3 suspension (nominally 5×10⁷ cells/ml)by means of a hemacytometer counting chamber and a microscope and inmost cases by means of a Coulter Counter. A final dilution to 1×10⁷cells/ml was made based on the averaged count (normally about 500-600cells were counted to obtain reliable statistics when the hemacytometermethod was employed). On day 1, solutions of the test compounds wereprepared and the mice injected with each mouse of a set of six micebeing injected with the same test compound at the same dosage level. Thedoses were based on the average weight of the animals (cage weights).

Also, on day 1 two types of controls were employed: (1) Normal (1 set):0.5 ml of the carrier used for the test compound; and (2) PositiveControl (1 set): cis-dichlorodiamineplatinum(II), a known anti-tumoragent, used at 7 or 8 mg/kg as a check on the response of the biologicaltest system.

The effectiveness of a compound was measured in terms of the % increasein life span (%ILS) of the test animals relative to the normal controls(calculated from the day of tumor inoculation (day 0)). In order tostandardize the test data and permit intercomparisons to be made, theday of evaluation was arbitrarily taken as that day corresponding totwice the mean life span (or average day of death) of the controls. Thisset a practical upper limit of 100% on the %ILS attainable. For purposesof calculation, survivors on the day of evaluation were considered tohave died on that day. The %ILS was formulated as: ##EQU1## ILS valuesin excess of 50% indicate significant anti-tumor activity, while valuesin excess of 75% indicate high activity.

The water-insoluble complexes of Examples 1 and 2 were tested asslurries in water containing the suspending agent Klucel (0.1-0.3%Klucel employed).

The data obtained from these tests are summarized in Table I.

                  TABLE I                                                         ______________________________________                                        Anti-Tumor Screening Data for the Complexes of Examples                       1 and 2 obtained on the S180a Tumor System                                    Complex                          Positive                                     of              Complex          Control.sup. (a)                             Example                                                                              Dose     %      30-day 60-day %    30-day                              #      (mg/kg)  ILS    survivors                                                                            survivors                                                                            ILS  survivors                           ______________________________________                                        1      10       99     5      0      63   0                                          20       52     2      0                                               1      10       62     1      0      60   0                                   2      20       62     4      2      63   0                                          40       -65    0      0                                                      80       -86    0      0                                                      160      -87    0      0                                               2      2.5       6     0      0      71   1                                           5       30     1      1                                                      10       78     4      3                                                      20       73       5.sup.(b)                                                                          4                                               ______________________________________                                         .sup.(a) Positive control = cis [Pt(NH.sub.3).sub.2 Cl.sub.2 ] 8 mg/kg in     saline.                                                                       .sup.(b) All tumor free.                                                 

Based on the data set forth in Table I, both the complex of Example 1,[{Pt(en)}₂ P₂ O₇ ], and the complex of Example 2, [{Pt(DAC)}₂ P₂ O₇ ]appeared to be effective against the S180a tumor screening system. Thecomplex of Example 1 was active at doses of 10 and 20 mg/kg; the toxicdose was not reached. The complex of Example 2 was also active at dosesof 10 and 20 mg/kg; it appeared toxic at doses of 40 mg/kg and greater.

EXAMPLE 4 Evaluation of Anti-Tumor Activity in the L1210, P388 and B16Tumor Systems

The complex of Example 1 was also screened for activity against mouselymphoid leukemia L210, mouse lyphocytic leukemia P388 and mousemelanoma B16. The complex of Example 2 was screened against mouselymphoid leukemia L1210. In these tests, the mean survival time of thetreated mice as compared with that of the control mice (T/C) wasdetermined. The T/C* was calculated as follows: ##EQU2## T/C values of125 or more represent significant anti-tumor activity.

The results of these tests are summarized in Table II.

                  TABLE II                                                        ______________________________________                                        Anti-Tumor Screening Data Results from the                                    B16, L1210 and P388 Tumor Systems                                             Complex.sup.c of                                                                       Tumor    Dose     Dose Range                                                                             Highest T/C                               Example #                                                                              System   Schedule (mg/kg)  (dose mg/kg)                              ______________________________________                                        1        B16      a        0.78-12.5                                                                              149(6.25)                                          B16      a        2.66-9.00                                                                              144(4.00)                                          L1210    b        1.56-25.0                                                                              125(12.5)                                          L1210    b        0.75-24.0                                                                              125(6.00)                                          P388     a        0.78-12.5                                                                              220(6.25)                                          P388     a        0.78-12.5                                                                              171(3.13)                                 2        L1210    b        12.5-200 190(12.5)                                          L1210    b        1.56-50.0                                                                              140(6.25)                                 ______________________________________                                         a 9 Daily doses on Days 1-9.                                                  b 3 doses on Days 1, 5, 9.                                                    c cThe carrier for all samples was saline with Tween80 used as a              suspending agent.                                                        

Based on the data set forth in Table II, it appears that the complex ofExample 1 was reproducibly active against all three of the tumor systemsthat it was tested against. The complex of Example 2 appeared to showexcellent activity against the L1210 tumor system.

What is claimed is:
 1. An amine platinum(II) pyrophosphate complex ofthe formula:

    [{Pt(A)}.sub.2 P.sub.2 O.sub.7 ]

wherein A is a diamine of the formula: NR¹ R² --CHR³ --CHR⁴ --NR⁵ R⁶ inwhich R¹ -R⁶, taken separately, are selected from hydrogen or loweralkyl and R³ and R⁴, taken together, represent the divalent radical:--CHR⁷ --CHR⁸ --CHR⁹ --CHR¹⁰ -- wherein R⁷ -R¹⁰ are selected from thegroup consisting of hydrogen and lower alkyl.
 2. The complex accordingto claim 1 wherein R¹ -R⁶, taken separately, represent hydrogen or loweralkyl.
 3. The complex according to claim 1 wherein each of R¹ -R⁶ ishydrogen.
 4. The complex according to claim 1 wherein R¹, R², R⁵ and R⁶are selected from hydrogen or lower alkyl and R³ and R⁴, taken together,represent the divalent radical: --CHR⁷ --CHR⁸ --CHR⁹ --CHR¹⁰ -- whereinR⁷ -R¹⁰ are selected from the group consisting of hydrogen and loweralkyl.
 5. The complex according to claim 4 wherein R¹, R², R⁵ and R⁶ arehydrogen and R³ and R⁴, taken together, represent the divalent radical:--CH₂ --CH₂ --CH₂ --CH₂ --.